CN114316012A - Antibacterial infection immune protein for koi and application - Google Patents

Abstract

The invention discloses an immune protein for resisting bacterial infection for koi and application thereof. The invention discloses an immune protein for resisting bacterial infection of koi, which is a protein with an amino acid sequence of SEQ ID NO.1. Experiments prove that the immunoprotein and the coding gene thereof can eliminate pathogenic bacteria in fish bodies and improve the survival rate: after the plasmid expressing the immune protein is injected into the body, the pathogenic bacteria in the fish body infected with the pathogenic bacteria are obviously lower than the control injected with the plasmid not expressing IPR, and the survival rate of the former is obviously higher than that of the latter. The immunoprotein and the coding gene thereof can be used for improving the immunity of the fish and further can be used for treating and preventing the pathogenic bacteria infection of the fish.

Description

Antibacterial infection immune protein for koi and application

Technical Field

The invention relates to an immune protein for resisting bacterial infection for koi and application thereof, belonging to the technical field of biology.

Background

The fancy carp is called as a swimming artwork by the body, gorgeous color and gorgeous pattern of the fancy carp, and is one of the most valuable ornamental fish varieties in the world in China. Different from edible carps, the fancy carps are subjected to multiple sorting in different growth stages, the operations of pulling a net, fishing and the like can cause the stress reaction of the fancy carps, so that the immunity is reduced, various pathogenic microorganisms are easy to breed, and the diseases are frequent. The diseases of koi are mostly caused by bacteria and parasites, and there are also a few diseases caused by viruses. However, the combined infection of bacteria has a more serious influence on the survival of koi, regardless of the cause of the disease. The antibiotic and the bactericide are mainly used by amateurs and fancy carp fans for treatment, but the treatment effect is poor due to the drug resistance of pathogenic bacteria to the antibiotic, and in addition, the use of the antibiotic causes pollution to water bodies and influences the health of fishes and human beings. In addition, for ornamental fish, although the fish diseases are cured, the fish diseases can leave shape defects, such as difficult regeneration after the fin is canker, and the original color of the broken scales is lost after the scales are regenerated. Therefore, the principle of 'prevention over treatment' for ornamental fishes is particularly important, and a new idea is provided for healthy breeding and disease prevention and control of fancy carps based on development of immune epidemic prevention products for improving the immune disease resistance level of the fancy carps.

Disclosure of Invention

The invention aims to solve the technical problems of improving the immunity of fish and resisting the infection of pathogenic bacteria.

In order to solve the technical problems, the invention firstly provides the application of the koi immune-related protein in the preparation of any functional product as follows:

D1) improving the immunity of the fish;

D2) treating and/or preventing disease in fish caused by infection with pathogenic bacteria;

D3) inhibiting the growth of pathogenic bacteria in the fish body;

D4) pathogenic bacteria in the fish body are eliminated;

D5) combating infestation of fish by pathogenic bacteria;

D6) protecting fish from pathogenic bacteria infection;

D7) the survival rate of fish infected by pathogenic bacteria is improved;

the koi immune-related protein is derived from koi (Cyprinus carpio koi) and is named IPR (IPR is A1), A2) or A3):

A1) a protein having an amino acid sequence of SEQ ID No. 1;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown by SEQ ID NO.1 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate the purification of the protein of A1), the amino terminus or the carboxy terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence Listing may be attached with the tags shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein A2) is a protein having identity of 75% or more than 75% with the amino acid sequence of the protein shown in SEQ ID NO.1 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The protein of A2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of A2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in SEQ ID NO.2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in SEQ ID NO.2 codes the protein shown in SEQ ID NO.1.

The invention also provides any one of the following uses of IPR-related biomaterials:

D1) improving the immunity of the fish;

D2) treating and/or preventing disease in fish caused by infection with pathogenic bacteria;

D3) inhibiting the growth of pathogenic bacteria in the fish body;

D4) pathogenic bacteria in the fish body are eliminated;

D5) combating infestation of fish by pathogenic bacteria;

D6) protecting fish from pathogenic bacteria infection;

D7) the survival rate of fish infected by pathogenic bacteria is improved;

the biomaterial is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding an IPR;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a cell line comprising B1) the nucleic acid molecule or a cell line comprising B2) the expression cassette.

In the above application, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) as follows:

b11) the coding sequence is cDNA molecule or DNA molecule of SEQ ID NO.2 in the sequence table;

b12) DNA molecule shown as SEQ ID NO.2 in the sequence table;

b13) a cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) and encoding an IPR;

b14) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b11) or b12) or b13) and encodes an IPR.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the IPR protein of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the isolated IPR protein of the present invention are derived from and identical to the nucleotide sequence of the present invention as long as they encode the IPR protein and have the function of the IPR protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID NO.1. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the expression cassette containing a nucleic acid molecule encoding an IPR protein (IPR gene expression cassette) according to B2) refers to a DNA capable of expressing an IPR protein in a host cell, and the DNA may include not only a promoter which initiates transcription of the IPR gene but also a terminator which terminates transcription of the IPR gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the IPR gene expression cassette can be constructed by using the existing expression vector.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid may be specifically pcdna3.1.

B3) The recombinant vector can be pCDNA3.1-IRP. The pCDNA3.1-IRP is a recombinant vector obtained by replacing a DNA fragment between HindIII and XhoI recognition sequences of pCDNA3.1 with a DNA fragment shown by SEQ ID NO.2 in a sequence table. pCDNA3.1-IRP can express fusion protein of IRP shown in SEQ ID NO.1 and 6 × His tag.

In the above application, the microorganism may be yeast, bacteria, algae or fungi.

In the above application, the cell line does not comprise propagation material.

IPR or said biological material, also belong to the scope of protection of the present aspect.

In the invention, the immunity can be the immunity of the fish to pathogenic bacteria.

The pathogenic bacteria may be bacteria of the genus Aeromonas (Aeromonas).

Further, the bacterium of the genus Aeromonas (Aeromonas) may be Aeromonas veronii (Aeromonas veronii) or Aeromonas hydrophila (Aeromonas hydrophila). In one embodiment of the present invention, Aeromonas veronii (Aeromonas veronii) is Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 (China general microbiological culture Collection center (CGMCC, Strain No. 1.927.) in one embodiment of the present invention, Aeromonas hydrophila (Aeromonas hydrophila) NX830 (national aquatic animal pathogen Bank, Collection No. BYK 20130805).

The fish may be E1, E2, E3 or E4:

e1, cyprinid;

e2, carp;

e3, carp;

e4, Koi (Cyprinus carpio koi).

In the present invention, the fish body may be in the spleen of a fish.

Experiments prove that the IPR and the coding gene thereof can clear pathogenic bacteria in fish bodies and improve the survival rate: after the plasmid expressing IPR is injected into the body, the pathogenic bacteria in the fish body infected with the pathogenic bacteria are obviously lower than the control injected with the plasmid not expressing IPR, and the survival rate of the former is obviously higher than that of the latter. The IPR and the coding gene thereof can be used for improving the immunity of the fish and further can be used for treating and preventing the pathogenic bacteria infection of the fish.

Drawings

FIG. 1 shows the expression level of Koi IRP gene in different tissues of healthy Koi. There was no significant difference in gene expression between tissues labeled with the same letter, and there was a significant difference in gene expression between tissues labeled with different letters.

FIG. 2 shows the expression change level of IRP gene in spleen tissue of Koi after Aeromonas veronii infection. PBS was used as control group, A.v was used as challenge group. Indicates that the difference reaches a significant level p < 0.05.

FIG. 3 shows the expression change level of IRP gene in Koi head and kidney tissue after Aeromonas veronii infection. A.v denotes the challenge group and PBS denotes the control group. Indicates that the difference reaches a significant level p < 0.05.

FIG. 4 shows the survival rate of Koi infected with Aeromonas veronii after injection of Koi pcDNA3.1-IRP and control plasmid pcDNA3.1. pCDNA3.1 is a control group, and IPR is a test group.

FIG. 5 variation of the expression level of inflammation-related factor. pCDNA3.1 is a control group, and IPR is a test group. Indicates that the difference reaches a significant level p < 0.05.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 in the following examples is a China general microbiological culture Collection center (CGMCC) strain with the strain number: 1.927, the preservation date is 9/1975. The website of the strain is as follows:

http://www.cgmcc.net/directory/detailcgmccid＝1.927&number＝1.927&genus＝&species＝&yiming＝&page＝1。

examples 1,

Discovery of IRP Gene

The inventor discovers a gene with obviously up-regulated expression (about 16 times) by performing transcriptome sequencing on spleen of fancy carp which develops after the infection of Aeromonas veronii, indicates that the gene is probably related to the immunity of the fancy carp, and marks the gene as IRP gene. In koi, the coding sequence of the IRP gene is shown as SEQ ID NO.2, and has 387 nucleotides in total, the amino acid sequence of the coding protein is shown as SEQ ID NO.1, and the signal peptide is not contained.

The nucleotide sequence of ORF of IRP gene (SEQ ID NO.2) is as follows:

ATGGTGTTCACCATAAAGGACATGAGCTTTAAGGCCGGGATGGAGATGAAGGTCACTGGAAAGACTAAACCAGGCTGTGAACAGTTCTCCATCAACATCGGTCACAACACCGACGCAATCGCTCTTCACTTCAACCCTCGCTTTAGCAGCAACGTCATCGTGTGCAACTCCAACCAGGGCGGCTGGGGAGCCGAGCATCACGAATCCTGTTTCCCCTTTCAACAGGGCGAAGAGTTCAAGCTGAGCATCACCTTCAACAACGACACCTTCTACATCAAGCTCCCGGAGGGCACCATGATGAGCTTCCCCAACCGCTTCGGCGACGACGTCTTCAAACACGTGCACGTGTCGGGAGACGTGAAGATCACCAGCATCAAGGTCAACTGA。

the IRP amino acid sequence (SEQ ID NO.1) is as follows:

MVFTIKDMSFKAGMEMKVTGKTKPGCEQFSINIGHNTDAIALHFNPRFSSNVIVCNSNQGGWGAEHHESCFPFQQGEEFKLSITFNNDTFYIKLPEGTMMSFPNRFGDDVFKHVHVSGDVKITSIKVN。

tissue expression distribution of koi IRP

And detecting the relative expression quantity of the IRP gene in different tissues in the healthy koi body by adopting a real-time fluorescent quantitative PCR method, and researching the tissue distribution of the IRP mRNA.

The specific operation method comprises the following steps: randomly selecting 12 tissues of healthy koi (with the weight of about 20g), wherein the tissues are as follows: gill, eye, head kidney, spleen, kidney, heart, muscle, skin, liver, blood, brain, and intestine. Total tissue RNA was extracted using RNAioso Plus (Takara 9109) and reverse transcribed to cDNA using reverse transcription kit (Takara RR 047A).

Primers for fluorescent quantitative PCR were designed based on the cDNA sequence of IRP, and the primer sequences were as follows:

qGal2F：5’-TGGAGATGAAGGTCACTGGAAAG-3’；

qGal2R：5’-TTGCTGCTAAAGCGAGGGTT-3’；

the primer sequences of the internal reference gene 40S ribosomal protein S11 gene are as follows:

qS11F：5’-CCGTGGGTGACATCGTTACA-3’；

qS11R：5’-TCAGGACATTGAACCTCACTGTCT-3’。

and detecting the relative expression quantity of the IRP in the 12 tissues by adopting an ABI7500 real-time fluorescence quantitative PCR instrument. The reaction conditions are as follows: at 95 ℃ for 30 s; 95 ℃ for 5s, 59.6 ℃ for 30s, 72 ℃ for 30s, 40 cycles.

As a result, as shown in FIG. 1, the IRP gene was expressed in all of the 12 tissues, and the expression level was the highest in the intestine, and significantly different from the expression levels in the other tissues (P < 0.05), and further, spleen, head kidney and kidney were observed. The intestines, the spleen and the head kidney are all main immune organs, and the fact that the gene is closely related to the immune function of koi is shown.

Expression change of koi IRP gene after infection of Aeromonas veronii

Taking Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 out of an ultra-low temperature refrigerator, streaking on an LB plate, culturing in an incubator at 28 ℃, recovering the strain, and selecting a single colonyEnlarging culture in LB liquid culture medium, centrifuging to collect thallus, resuspending with PBS, adjusting to 5 × 10⁶CFU/mL to obtain suspension of Aeromonas veronii.

Randomly selecting 60 healthy fancy carps (about 20g), and randomly and averagely dividing into 2 groups, a toxicity counteracting group and a control group, wherein each group comprises 3 parallel cylinders. 100 mu L of Aeromonas veronii suspension is injected into the abdominal cavity of each fancy carp in the toxicity counteracting group, and 100 mu L of LPBS is injected into the abdominal cavity of each fancy carp in the control group. Randomly selecting 6 brocarded carps from the challenge group and the control group respectively before (0h) infection and 6h,12h,24h,48h,96h and 7d after infection, collecting spleen and head kidney tissues, placing in liquid nitrogen for quick freezing, storing at-80 ℃ for RNA extraction, and mixing every 2 fishes to reduce individual errors. And detecting the relative expression quantity of the koi IRP gene by adopting a real-time fluorescent quantitative PCR method.

The results (fig. 2, 3) show that at 12h after infection by aeromonas veronii, the koi IRP gene was significantly up-regulated in spleen tissue expression and then decreased below the control group; in the head and kidney tissues, the gene is obviously up-regulated from 12 hours after bacterial infection until 96 hours, and the expression level is still obviously higher than that of a control group (P is less than 0.05), so that the IRP gene is mainly responded and obviously up-regulated in the head and kidney tissues after the Koi is infected with Aeromonas veronii, and plays an immune regulation role.

Construction of koi IRP eukaryotic expression plasmid

Designing amplification primers F respectively carrying HindIII and XhoI enzyme cutting sites according to the mRNA sequence of the koi IRP gene in the sequencing result (SEQ ID NO.3: CC)AAGCTTGGGATGGTGTTCACCATAAAGGACAT) and R (SEQ ID NO.4: CC)CTCGAGTCAGTGGTGGTGGTGGTGGTGGTTGACCTTGATGCT). Taking spleen cDNA of koi (Cyprinus carpio koi) as a template, and taking F and R as primers to carry out PCR amplification, thereby obtaining a DNA fragment containing an IRP gene coding sequence.

The eukaryotic expression vector pCDNA3.1(Invitrogen) is cut by restriction enzymes HindIII and XhoI, and a vector framework is obtained after recovery; the DNA fragment of the IRP gene coding sequence is cut by restriction enzymes HindIII and XhoI, the obtained fragment is connected with a vector framework, and the obtained recombinant vector with correct sequence is marked as pCDNA3.1-IRP.

pCDNA3.1-IRP is a recombinant vector obtained by replacing the DNA fragment between HindIII and XhoI recognition sequences of pCDNA3.1 with the DNA fragment shown in SEQ ID NO.2 of the sequence Listing. pCDNA3.1-IRP can express fusion protein of IRP shown in SEQ ID NO.1 and 6 × His tag.

Application of koi IRP eukaryotic expression plasmid

1. The effect of Cyprinus Carpio IRP on eliminating pathogenic bacteria Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 of Cyprinus Carpio

(1) Plasmid injection: pCDNA3.1-IRP was diluted to 200. mu.g/mL in PBS, which was the pCDNA3.1-IRP plasmid injection. The blank plasmid pCDNA3.1 was diluted to 200. mu.g/mL in PBS, the control plasmid injection. 8 fancy carps (about 20g) are randomly divided into 2 groups, each group has 4 tails, and the two groups are respectively a control group and a test group. Each fish of the test group was injected with 100. mu.L of pCDNA3.1-IRP plasmid injection, and each fish of the control group was injected with 100. mu.L of control plasmid injection.

(2) Preparation of pathogen suspension: culturing Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 in LB medium to OD600 of 0.6-0.8, centrifuging (8000g, 2min), pouring supernatant, suspending the thallus in PBS, adjusting to final concentration of 5 × 10⁶CFU/mL, namely the Aeromonas veronii suspension.

(3) Infection with offensive toxin: at 72 hours after the plasmid injection in step (1), 100. mu.L of Aeromonas veronii suspension of the above-mentioned (2) was injected into each fish of the control group and the test group. At 24h post-infection, koi was anesthetized with MS-222, dissected, spleen tissue removed, and weighed. Sterilized PBS was added at 10. mu.L/mg, ground with a sterile grinding bar, 100. mu.L of spleen homogenate was spread on LB plates, 2 plates were applied per sample to obtain an average, and colony counting was performed after the plates were incubated for 24 hours at 28 ℃ in an incubator, and statistical analysis was performed. As a result, as shown in Table 1, the number of colonies in the spleen of koi in the test group (16 colonies/mg spleen) was significantly lower (P < 0.05) than that in the spleen of koi in the control group (44 colonies/mg spleen).

TABLE 1 spleen bacterial infection colony counts for fancy carp (counts/mg spleen)

2. Protective effect of koi IRP on koi against pathogenic bacteria Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 infection

(1) Plasmid injection: pCDNA3.1-IRP was diluted to 200. mu.g/mL in PBS, which was the pCDNA3.1-IRP plasmid injection. The blank plasmid pCDNA3.1 was diluted to 200. mu.g/mL in PBS, the control plasmid injection. 16 fancy carps (about 20g) are randomly divided into 2 groups, each group has 8 tails, and the two groups are respectively a control group and a test group. Each fish of the test group was injected with 100. mu.L of pCDNA3.1-IRP plasmid injection, and each fish of the control group was injected with 100. mu.L of control plasmid injection.

(2) Preparation of pathogen suspension: culturing Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 in LB medium to OD600 of 0.6-0.8, centrifuging (8000g, 2min), pouring supernatant, suspending the thallus in PBS, adjusting to final concentration of 5 × 10⁷CFU/mL, namely the Aeromonas veronii suspension.

(3) At 72 hours after the plasmid injection in step (1), 100. mu.L of Aeromonas veronii suspension of the above-mentioned (2) was injected into each fish of the control group and the test group. And observing and counting the death conditions of all groups of fancy carps within 7d, fishing out the dead carps in time, and drawing a survival curve by using Graphpad.

The results (fig. 4) showed that the survival rate was higher (87.5%) for the test group at 7 days than that of the control group at 7 days (62.5%). The IRP is shown to be capable of enhancing the resistance of the fancy carp to infection of pathogenic bacteria.

3. Elaphe cyprinii IRP has effect in eliminating pathogenic bacteria Aeromonas hydrophila (Aeromonas hydrophylla, A.h) NX830 of Cyprinus carpioides

(1) Plasmid injection: pCDNA3.1-IRP was diluted to 200. mu.g/mL in PBS buffer, i.e., pCDNA3.1-IRP plasmid injection. The blank plasmid pCDNA3.1 is diluted to 200 mu g/mL in PBS buffer solution, and then the control plasmid injection is obtained. 6 brocade carps (about 30g) are randomly divided into 2 groups, each group has 3 carps, and the two groups are respectively a control group and a test group. Each fish in the test group was injected intramuscularly with 100. mu.L of plasmid injection (injected into the muscle rich area in front of dorsal fin root), and each fish in the control group was injected with 100. mu.L of control plasmid injection.

(2) Suspension of pathogenic bacteriaPreparation: culturing Aeromonas hydrophila (Aeromonas hydrophila, A.h) NX830 (national aquatic animal pathogen Bank, accession number: BYK20130805) to OD by LB culture medium₆₀₀0.6-0.8, centrifuging (8000g, 2min), pouring supernatant, suspending the thallus in PBS buffer solution to final concentration of 1 × 10⁸CFU/mL, namely the aeromonas hydrophila suspension.

(3) Infection with offensive toxin: at 72h after the plasmid injection in step (1), 100. mu.L of (2) suspension of Aeromonas hydrophila was injected into each fish of the control group and the test group. At 96h post-infection, koi was anesthetized with MS-222, dissected, spleen tissue removed, and weighed. Sterilized PBS buffer was added at 10. mu.L/mg, ground with a sterile grinding bar, 50. mu.L of spleen homogenate was spread on LB plates, 2 plates were applied per sample to obtain an average, and colony counting was performed after the plates were incubated for 24 hours at 28 ℃ in an incubator, and statistical analysis was performed. As a result, as shown in Table 2, the number of colonies in the spleen of koi in the test group (4 colonies/mg spleen) was significantly lower (P < 0.05) than that in the spleen of koi in the control group (81 colonies/mg spleen).

TABLE 2 spleen colony counts (counts/mg spleen) for brocade carp (30g)

4. Expression analysis of head and kidney tissue immune related gene after injection of koi IRP eukaryotic expression plasmid

pCDNA3.1-IRP was diluted to 200. mu.g/mL in PBS buffer, i.e., pCDNA3.1-IRP plasmid injection. The blank plasmid pCDNA3.1 is diluted to 200 mu g/mL in PBS buffer solution, and then the control plasmid injection is obtained. 20 brocade carps (about 30g) are randomly divided into 2 groups, 10 carps in each group, and the two groups are respectively a control group and a test group. Each fish in the test group was injected intramuscularly with 100. mu.L of plasmid injection (injected into the muscle rich area in front of dorsal fin root), and each fish in the control group was injected with 100. mu.L of control plasmid injection. At 72h after injection, 6 fishes in each group were randomly selected, immune tissue head and kidney were collected, total RNA was extracted, and reverse transcription was performed to cDNA. In order to reduce individual errors, 2 fishes are mixed, and the relative expression quantity of mRNA of several immune related genes is detected by a real-time fluorescence quantitative PCR method.

The results show (as shown in figure 5) that after the injection of IRP eukaryotic expression plasmid for 72h, the expression of proinflammatory factors TNF-alpha and IL-6 (respectively 1.61 times and 6.44 times of the control plasmid group) and the expression of complement system C4 (8.33 times of the control group) of the koi head and kidney tissues and the expression of the IL-10 (0.26 times of the control plasmid group) can be rapidly regulated up, and the expression of the inflammation suppressor factor can be reduced. The results suggest that IRP can promote inflammation in the head and kidney and activate the complement system to exert immunomodulatory effects.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> research institute of Water science in Beijing city (national research center for freshwater fishery engineering)

<120> antibacterial infection immune protein for koi and application

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 128

<212> PRT

<213> fancy carp (Cyprinus carpio Koi)

<400> 1

Met Val Phe Thr Ile Lys Asp Met Ser Phe Lys Ala Gly Met Glu Met

1 5 10 15

Lys Val Thr Gly Lys Thr Lys Pro Gly Cys Glu Gln Phe Ser Ile Asn

20 25 30

Ile Gly His Asn Thr Asp Ala Ile Ala Leu His Phe Asn Pro Arg Phe

35 40 45

Ser Ser Asn Val Ile Val Cys Asn Ser Asn Gln Gly Gly Trp Gly Ala

50 55 60

Glu His His Glu Ser Cys Phe Pro Phe Gln Gln Gly Glu Glu Phe Lys

65 70 75 80

Leu Ser Ile Thr Phe Asn Asn Asp Thr Phe Tyr Ile Lys Leu Pro Glu

85 90 95

Gly Thr Met Met Ser Phe Pro Asn Arg Phe Gly Asp Asp Val Phe Lys

100 105 110

His Val His Val Ser Gly Asp Val Lys Ile Thr Ser Ile Lys Val Asn

115 120 125

<210> 2

<211> 387

<212> DNA

<213> fancy carp (Cyprinus carpio Koi)

<400> 2

atggtgttca ccataaagga catgagcttt aaggccggga tggagatgaa ggtcactgga 60

aagactaaac caggctgtga acagttctcc atcaacatcg gtcacaacac cgacgcaatc 120

gctcttcact tcaaccctcg ctttagcagc aacgtcatcg tgtgcaactc caaccagggc 180

ggctggggag ccgagcatca cgaatcctgt ttcccctttc aacagggcga agagttcaag 240

ctgagcatca ccttcaacaa cgacaccttc tacatcaagc tcccggaggg caccatgatg 300

agcttcccca accgcttcgg cgacgacgtc ttcaaacacg tgcacgtgtc gggagacgtg 360

aagatcacca gcatcaaggt caactga 387

<210> 3

<211> 34

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

ccaagcttgg gatggtgttc accataaagg acat 34

<210> 4

<211> 44

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ccctcgagtc agtggtggtg gtggtggtgg ttgaccttga tgct 44

Claims (10)

1. The application of the koi immune-related protein in preparing any functional product comprises the following steps:

D1) improving the immunity of the fish;

D2) treating and/or preventing disease in fish caused by infection with pathogenic bacteria;

D3) inhibiting the growth of pathogenic bacteria in the fish body;

D4) pathogenic bacteria in the fish body are eliminated;

D5) combating infestation of fish by pathogenic bacteria;

D6) protecting fish from pathogenic bacteria infection;

D7) the survival rate of fish infected by pathogenic bacteria is improved;

the koi immune-related protein is A1), A2) or A3) as follows:

A1) a protein having an amino acid sequence of SEQ ID No. 1;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown by SEQ ID NO.1 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

2. Use according to claim 1, characterized in that: the immunity is immunity of the fish to pathogenic bacteria.

3. Use according to claim 1 or 2, characterized in that: the pathogenic bacteria are Aeromonas (Aeromonas) bacteria;

further, the bacterium belonging to the genus Aeromonas (Aeromonas) is Aeromonas veronii (Aeromonas veronii) or Aeromonas hydrophila (Aeromonas hydrophylla).

4. Use according to any one of claims 1 to 3, characterized in that: the fish is the following E1, E2, E3 or E4:

e1, cyprinid;

e2, carp;

e3, carp;

e4, Koi (Cyprinus carpio koi).

5. Use of a biological material related to koi immune-related protein according to claim 1, wherein the biological material is selected from the group consisting of:

D1) improving the immunity of the fish;

D2) treating and/or preventing disease in fish caused by infection with pathogenic bacteria;

D3) inhibiting the growth of pathogenic bacteria in the fish body;

D4) pathogenic bacteria in the fish body are eliminated;

D5) combating infestation of fish by pathogenic bacteria;

D6) protecting fish from pathogenic bacteria infection;

D7) the survival rate of fish infected by pathogenic bacteria is improved;

the biomaterial is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding a koi immune-related protein according to claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a cell line comprising B1) the nucleic acid molecule or a cell line comprising B2) the expression cassette.

6. Use according to claim 5, characterized in that: B1) the nucleic acid molecule is b11) or b12) or b13) or b14) as follows:

b11) the coding sequence is cDNA molecule or DNA molecule of SEQ ID NO.2 in the sequence table;

b12) DNA molecule shown as SEQ ID NO.2 in the sequence table;

b13) a cDNA molecule or a genomic DNA molecule having 75% or more identity with the nucleotide sequence defined in b11) or b12) and encoding a koi immune-related protein according to claim 1;

b14) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence defined by b11) or b12) or b13) under strict conditions and encodes the koi immune related protein in the claim 1.

7. Use according to claim 5 or 6, characterized in that: the immunity is immunity of the fish to pathogenic bacteria.

8. Use according to any one of claims 5 to 7, characterized in that: the pathogenic bacteria are Aeromonas (Aeromonas) bacteria;

further, the bacterium belonging to the genus Aeromonas (Aeromonas) is Aeromonas veronii (Aeromonas veronii) or Aeromonas hydrophila (Aeromonas hydrophylla).

9. Use according to any one of claims 5 to 8, characterized in that: the fish is the following E1, E2, E3 or E4:

e1, cyprinid;

e2, carp;

e3, carp;

e4, Koi (Cyprinus carpio koi).

10. A koi immune-related protein according to claim 1 or a biological material according to claim 5 or 6.

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